EP2549986A1 - Makrophagenabgabe an mehrere ziele - Google Patents

Makrophagenabgabe an mehrere ziele

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Publication number
EP2549986A1
EP2549986A1 EP11760255A EP11760255A EP2549986A1 EP 2549986 A1 EP2549986 A1 EP 2549986A1 EP 11760255 A EP11760255 A EP 11760255A EP 11760255 A EP11760255 A EP 11760255A EP 2549986 A1 EP2549986 A1 EP 2549986A1
Authority
EP
European Patent Office
Prior art keywords
agent
compartment
delivery system
target
sirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP11760255A
Other languages
English (en)
French (fr)
Other versions
EP2549986B1 (de
EP2549986A4 (de
Inventor
Mansoor M. Amiji
Mayur Kalariya
Shardool Jain
Husain Attarwala
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northeastern University Boston
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Northeastern University Boston
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Publication date
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Publication of EP2549986A1 publication Critical patent/EP2549986A1/de
Publication of EP2549986A4 publication Critical patent/EP2549986A4/de
Application granted granted Critical
Publication of EP2549986B1 publication Critical patent/EP2549986B1/de
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    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
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Definitions

  • This disclosure is in the field of medicine, and more specifically relates to a multi- compartmental nanoparticulate system for diagnosing, monitoring, and treating inflammation and/or disease. These systems are capable of targeting specific cell types, co-administration of incompatible materials, as well as temporal control of payload release.
  • Macrophages are important immune cells in the body that regulate different biological processes involved in inflammatory and infectious diseases. Macrophages originate from the mononuclear phagocyte system (MPS) in the bone marrow.
  • the MPS system is comprised of monocytes and tissue macrophages in their various forms.
  • the primary function of monocytes is phagocytosis.
  • monocytes Upon exiting the bone marrow, monocytes circulate in the blood and become activated in various tissues. Once the monocyte reaches the extravascular tissue, it matures into a larger phagocytic cell called a macrophage. They reside in the liver (Kupffer cells), lungs (alveolar macrophages), spleen, lymph nodes, thymus, gut, marrow, brain, connective tissue and serous cavities.
  • T-lymphocytes such as interferon-gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF-a) and granulocyte-monocyte colony stimulating factor (GM-CSF) can act as signals to activate macrophages.
  • IFN- ⁇ interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • GM-CSF granulocyte-monocyte colony stimulating factor
  • the activating signal can come from microbial products such as lipopolysaccharides, immune complexes, chemical mediators, and extracellular matrix proteins including fibronectin.
  • the instant disclosure is directed towards methods of imaging, diagnosing, and/or treating a disease or condition.
  • the multicompartmental delivery systems described herein allow for the administration of one or more agents such as, but not limited to, imaging agents, diagnostic agents, therapeutic agents, vaccines, proteins, gene constructs, and small molecules. These systems allow for co-administration of incompatible agents (i.e., hydrophobic and hydrophilic agents), as well as temporal control over the release of administered agents.
  • Figure 1 Differential interference contrast and fluorescence microscopy images of cellular uptake and green fluorescent protein (GFP) transfection in adherent J774A.1 murine alveolar macrophages using nanoparticles-in-emulsion (NiE) formulations.
  • the uptake studies were carried out with rhodamine-labeled gelatin nanoparticles in saffiower oil containing water- in-oil-in- water (W/O/W) multiple emulsion and the GFP transfection after 24 hours was observed with EGFP-N1 plasmid DNA-encapsulated gelatin nanoparticles in NiE
  • Figure 2 Illustrative examples of multi-compartmental delivery systems of the invention.
  • the formulations can be designed to have solid hydrophilic or hydrophobic nanoparticles in a larger solid microsphere (a), nanoparticles in water-in-oil-in- water multiple emulsions (b), or nanoparticles or oil droplets in a gel (c) , etc.
  • the combination of different constructs is based on the properties of the payload and the desired application of the system.
  • Figure 3 Schematic representation of the nanoparticles -in-microsphere ( M) delivery vehicle including scanning electron micrographs of small interfering RNA
  • siRNA A encapsulated type B gelatin nanoparticles and siRNA-containing MM.
  • siRNA-encapsulated gelatin nanoparticles had an average diameter of less than 300 nm with a narrow size distribution, and an encapsulation efficiency of approximately 90 ⁇ 5%.
  • siRNA encapsulated MM had an average diameter of 2.4 ⁇ 0.9 IJm with a narrow size distribution. Encapsulating efficiency of siRNA decreased to approximately 55 ⁇ 3% in MM.
  • FIG. 4 The diagram above depicts the key functions of a macrophage cell.
  • Panel A shows the ability of macrophages to detect the presence of bacteria (pathogen) via specific receptors (pathogen recognition receptors or toll like receptors (TLR)) on its surface, which leads to a cascade of the down-stream signals that ultimately lead to release of cytokines and chemokines to signal other cells about bacterial invasion.
  • Panel B highlights the ability of these cells to phagocytose, either via non-specific or receptor- mediated endocytosis, and degrades the bacteria as seen in the phagolysosomal compartment. The degraded bacterial fragments can be excreted out of the cell via process called exocytosis.
  • the degraded antigenic peptide products can be displayed at the cell surface with the help of class II MHC molecule. This process helps in communicating with the T-helper cells.
  • naive T cell requires a secondary signal for activation, which is provided by interactions between B7 receptor on macrophages and CD28 ligand molecule on T-cell. Upon activation, these cells can further lead to generation of Thl and Th2 immune response.
  • macrophages are also capable of releasing cytokines such as IL-6, 12 that can influence adaptive immune response.
  • IL-6 nuclear Factor- ⁇
  • ⁇ Inhibitor of NF- ⁇ IL-6-Interleukin-6
  • IL-12-Interleukin-12 IL-12-Interleukin-12.
  • Figure 5 Different functions of tumor associated macrophages in promoting tumor growth and metastases.
  • Figure 6 Schematic illustration for the preparation of tuftsin-modified macrophage- targeted alginate nanoparticles for plasmid DNA encapsulation and delivery.
  • Figure 7 Time-dependent fluorescence microscopy images of the untreated cells and cells treated with unmodified, scrambled peptide -modified, and tuftsin-modified rhodamine- dextran encapsulated alginate nanoparticles. The images were taken at 15, 30, and 60 minutes post-administration and the original magnification was 40X.
  • Figure 8 Time-dependent quantitative analysis of GFP expression in J774A.1 macrophage cell line upon administration of EGFP-Nl plasmid DNA in control and tuftsin- modified alginate nanoparticles.
  • Figure 10 Bright-field and fluorescent microscopy images of NiB (A&B) and ME (C&D) showing stable non-leaky multiple emulsion globules of 5.0 ⁇ or less diameter.
  • Figure 11 Differential interference contrast and fluorescent microscopy images showing green fluorescent protein expression of in J774A.1 cells 24 hour post-treatment with EGFP-Nl plasmid DNA complexed with Lipofectin® (A,B), or encapsulated in NiE (C,D), ME (E, F) and GNP (G,H). Original magnification was 40X.
  • FIG. 12 Quantitative green fluorescent protein (GFP) expression by ELISA showing transgene expression with EGFP-Nl plasmid DNA encapsulated in GNP, ME, and NiB as well as complexed with Lipofectin®. Highest levels of GFP expression was observed with- NiE formulation relative to all other tested.
  • GFP green fluorescent protein
  • FIG. 13 Formulation of siRNA-encapsulated nanoparticles and NiMOS.
  • siRNA small interfering RNA
  • Lane I 10 bp ladder; lane 2: naked siRNA; lane 3: naked siRNA treated with protease; lane 4: naked siRNA treated with protease and RNAse A; lane 5: siRNA extracted from gelatin nanoparticles; lane 6: siRNA extracted from gelatin nanoparticles and then treated with RNAse A; lane 7: siRNA extracted from NiMOS; lane 8:.
  • the siRNA-encapsulated gelatin nanoparticles had an average diameter of less than 300 nm with a narrow size distribution, and an encapsulation efficiency of approximately 90: 1 : 5%.
  • siRNA - encapsulated NiMOS had an average diameter of 2.4: 1 : 0.9um with a narrow size distribution. Encapsulating efficiency of siRNA decreased to approximately 55 ⁇ 3% in NiMOS.
  • Figure 14 Dextran sulfate sodium-induced acute colitis murine model, a) Percent weight loss in 6-8 weeks old female Balb/c mice fed with 3.5% (w/w) dextran sulfate sodium (DSS) to induce acute colitis in comparison to weight of the control mice fed with regular drinking water. Dips in the curve body correspond to weight measurements immediately following the fasting periods, b) Tissue myeloperoxidase activity per mg total protein content in the same group of mice, c) Bright-field images of H&E stained sections of the colon harvested from each group. Images are shown at lOx and 40x of original magnifications. Sections from the control group exhibit features normal, and healthy colon tissue. Intestinal tissues from the DSS control group showed a significant level of infiltration of white blood cells, abnormal mucosal architecture and goblet cell depletion.
  • DSS dextran sulfate sodium
  • Control naive, no colitis
  • DSS Control colitis, no treatment
  • Blank NiMOS colitis, blank microspheres
  • TNF-a NiMOS colitis, TNF-a siRNA containing microspheres
  • Scramble NiMOS colitis, scramble siRNA containing microspheres.
  • Figure 16 Cytokine and chemokine profiles in the GI tract.
  • the cytokine expression profile upon administration of three doses of NiMOS (blank or encapsulating TNF-a siRNA, scramble siRNA) to animals under continuous dextran sodium sulfate (DSS) exposure was determined using a chemiluminescent ELISA Q-PlexTM Mouse Cytokine Screen (Quansys Biosciences, Logan, UT).
  • Interleukin 6 Interleukin 6
  • IL-12 p70 Interleukin 12 p70
  • IL-7 Interleukin 7
  • levels of pro-inflammatory cytokines are low in the healthy control group at both time points as compared to inflamed tissue. The highest concentration of pro-inflammatory cytokines was observed in animals treated with blank or scrambled siRNA containing NiMOS. Levels were lowest in groups receiving TNF-a silencing siRNA.
  • Granulocyte macrophage colony-stimulating factor (GMCSF); monocyte chemotactic protein-I (MCP-I); monocyte inflammatory protein I alpha ( ⁇ - ⁇ ). Values expressed as mean ⁇ S.D. (n 4).
  • FIG. 17 Evaluation of colonic tissue histopathology. Images of the stained sections of the colon harvested from each group at days post-administration. Images of tissue cryosections obtained on day 10 and day 14 of the study are shown at magnifications of lOx and 40x of the original size. Sections from the control group show normal and healthy colon tissue. Intestinal tissues from the dextran sulfate sodium (DSS) control group, the group treated with blank NiMOS as well as NiMOS encapsulating scrambled TNF-a siRNA sequence showed a significant level of infiltration of white blood cells, abnormal mucosal architecture and a certain degree of goblet cell depletion. Tissue from the group receiving TNF-a silencing NiMOS exhibited a tissue structure more closely resembling that of healthy tissue.
  • DSS dextran sulfate sodium
  • Figure 19 Bright- field and fluorescent microscopy images of NiE (A&B) and ME (C&D) showing stable non-leaky multiple emulsion globules of 5.0 um or less diameter. Original magnification was 60X.
  • Figure 20 Determination of EGFP-N1 plasmid DNA stability by agarose gel electrophoresis.
  • Figure 21 Bright-field and fluorescent images in figure (A,B), (C,D) and (E,F) shows cellular uptake of Rhodamine dextran containing ME formulation by J774A.1 cells at 60 minutes, 90 minutes and 120 minutes time points respectively.
  • Bright field and fluorescent images in figure (G,H), (I, J) and (K,L) shows cellular uptake of Rhodamine dextran (70,000 MW) containing NiE formulation by J774A.1 cells at 60 minutes, 90 minutes and 120 minutes time points respectively.
  • Original magnification was 40x.
  • Figure 22 Fluorescent microscopy images showing localization of DNA (green), GNP (red) and Nucleus (blue) within J774A.1 cells at 30 minutes, 2 hours and 6 hours time points post-treatment with either NiE, ME or GNP formulations. Original magnification was [0031]
  • Figure 23 Evaluation of qualitative gene transfection by fluorescent microscopy: (a) Differential interference contrast and fluorescent microscopy images showing green fluorescent protein expression of in J774A.1 cells 24 hour post-treatment with EGFP-Nl plasmid DNA complexed with Lipofectin ® (A,B), or encapsulated in NiE (C,D), ME (E,F) and GNP (G,H).
  • FIG. 24 Quantitative green fluorescent protein (GFP) expression by ELISA showing transgene expression with EGFP-Nl plasmid DNA encapsulated in GNP, ME, and NiE as well as complexed with Lipofectin®. Highest levels of GFP expression was observed with NiE formulation relative to all other tested.
  • GFP green fluorescent protein
  • Figure 25 (A) Agarose gel electrophoresis showing intact plasmid of approximately 3.7 kilo base pairs (lane 2). Lane 4 shows presence of a DNA fragment of approximately 500bps in size correlating with size of IL-10 transgene in the plasmid vector. (B) mIL-10 plasmid constructs.
  • Figure 26 Qualitative IL- 10 mRNA expression by RT-PCR in J774A.1 cells. NiE formulations showed higher mIL-10 transgene transcript expression relative to all other tested.
  • Figure 27 Quantitative IL-10 protein expression by ELISA showing transgene expression with mIL-10 plasmid DNA encapsulated in GNP, ME, and NiE as well as complexed with Lipofectin®. Highest levels of mILlO expression were observed with NiE formulation relative to all other tested.
  • Figure 28 RT-PCR analysis for TNFa and IL1 ⁇ mRNA in untreated cells (lane 3) or treated with naked plasmid (lane 4), mILlO plasmid complexed with Lipofectin® (lane 5), mILlO plasmid in NiE (lane 6), mILlO plasmid in ME (lane 7) or mILlO plasmid in GNP (lane 8) at 24 hour and 48 hour time points after LPS stimulation.
  • Figure 29 Quantitative TNFa protein expression by ELISA showing IL-10 transfection mediated suppression of TNFa in either untreated J774A.1 cells or J774A.1 cells transfected with mIL-10 containing NiE, ME, GNP, Lipofectin® or naked mIL-10 plasmid.
  • Figure 30 Quantitative ILip protein expression by ELISA showing IL-10 transfection mediated suppression of IL1 ⁇ in either untreated J774A.1 cells or J774A.1 cells treated with mIL-10 containing NiE, ME, GNP, Lipofectin or naked mIL-10 plasmid.
  • This disclosure is related in part to a composition of matter for safe and efficient delivery of image contrast agents, drugs, genes, small interfering RNA (siRNA), and vaccination molecules to macrophages upon administration in the body by oral, nasal, inhalation, injection or any other route known to those skilled in the art.
  • image contrast agents drugs, genes, small interfering RNA (siRNA), and vaccination molecules to macrophages upon administration in the body by oral, nasal, inhalation, injection or any other route known to those skilled in the art.
  • the instant disclosure is directed towards a multicompartment delivery system comprising one or more inner aqueous compartments encapsulated within an outer hydrophobic compartment, the outer hydrophobic compartment being surrounded by an exterior aqueous compartment.
  • a first agent encapsulated within the one or more inner aqueous compartments.
  • the first agent is selected from the group consisting of an imaging agent, a diagnostic agent, a therapeutic agent, a vaccine, a protein, or a gene construct.
  • a second agent is encapsulated within the outer hydrophobic compartment.
  • Encapsulated is defined herein to include dissolved in a material, associated with a material, meaning that an agent or nanoparticle can be on the surface, within the material, or surrounded by the material that comprises the compartment.
  • the second agent can be selected from the group consisting of an imaging agent, a diagnostic agent, a therapeutic agent, a vaccine, a protein, or a gene construct.
  • the multicompartment delivery system disclosed herein can also comprise an outer compartment that forms a microsphere. Methods of producing a microsphere are known in the art and include the single emulsion method and solvent displacement method (see, e.g., Furtado Mosqueira et al.
  • the microsphere can be about 0.5 to 500 microns in size.
  • the inner aqueous compartment is about 50 nm to about 300 nm.
  • the inner aqueous compartment comprises nanoparticles.
  • the nanoparticles comprise gelatin or alginate.
  • the outer hydrophobic compartment comprises safflower oil.
  • one or more target-specific binding agents are attached to the surface of the outer hydrophobic compartment.
  • the one or more target-specific binding agents attached to the surface of the outer hydrophobic compartment is tuftsin. Methods of attaching binding agents to a particle or carrier are known in the art (see, e.g., Magadala et al. AAPS J. (2008) 10(4): 565-576).
  • the exterior aqueous compartment comprises water.
  • the multicompartment delivery system further comprising one or more target-specific binding agents attached to the surface of the inner aqueous compartment or the outer hydrophobic compartment.
  • one or more target-specific binding agents are attached to the inner aqueous compartment and the outer hydrophobic compartment.
  • Further embodiments further comprise one or more target-specific binding agents attached to the inner aqueous compartment, direct the inner compartment to an intracellular location and the one or more target-specific binding agents attached to the outer hydrophobic compartment direct the microsphere to a cell.
  • the delivery vehicles disclosed herein are specifically designed to have multiple compartments (multicompartment) to afford the following two advantages: (1) ability to sequester the payload in each of the compartment due to incompatibility (e.g., poor solubility), instability, or desire for temporal-controlled release and delivery; and (2) ability to overcome the different tissue, cell, and intracellular barriers to afford efficient delivery of the payload to the desired biological target site. This is especially critical for nucleic acid-based therapeutics such as genes, oligonucleotides, and siRNA.
  • the multi-compartmental delivery systems are designed to meet the needs of the payload (e.g., hydrophobic versus hydrophilic), the site of delivery in the body, the intended application (e.g., drugs versus vaccines), and the disease of interest.
  • the multicompartment delivery vehicles disclosed herein can be nanoparticles-in- microspheres (Figure 3). Nanoparticles-in-microspheres can have an outer compartment ⁇ i.e., microsphere). In certain embodiments, the microsphere is a "solid.” As used herein, the term “solid” means a substance that is structurally rigid and resistant to changes in shape. Such microspheres are composed of materials such as poly(epsilon-caprolactone), (PCL), poly(D,L- lactide-co-glycolide) (PLGA), and polymethylmethacrylate. The microspheres can be from about 500 nanometers to about 1.5 microns.
  • the microspheres encapsulate solid nanoparticles that form an inner compartment.
  • the nanoparticles can be composed of a material different from the microsphere.
  • the nanoparticles that are composed of hydrophilic materials are encapsulated within microspheres composed of hydrophobic materials.
  • hydrophobic nanoparticles are encapsulated within hydrophilic microspheres
  • the hydrophobic microsphere can be composed of an oil such as safflower oil or any other biologically compatible oil, a wax or hydrophobic molecules such as poly(epsilon-caprolactone), (PCL), poly(D,L- lactide-co-glycolide) (PLGA), and polymethylmethacrylate.
  • nanomaterials can be obtained from Phosphorex Inc. (Fall River, MA).
  • methods of making nanoparticles are known in the art (see, e.g., Yang et al, Int. J. Pharmaceutics (2006) 324 (2): 185-190); Gou et al, Int. J. Pharmaceutics (2009) 375 (1-2): 170-176).
  • the Yang et al, Int. J. Pharmaceutics (2006) 324 (2): 185-190 Gou et al, Int. J. Pharmaceutics (2009) 375 (1-2): 170-176.
  • nanoparticles can be from about 20 nanometers ("nm") to about 300 nanometers; from about 20 nm to about 30 nm; from about 30 nm to about 40 nm; from about 40 nm to about 50 nm; from 50 nm to about 100 nm; from about 100 nm to about 150 nm; from about 150 nm to about 200 nm; from about 200 nm to about 250 nm; and from about 250 to about 300 nm.
  • nm nanometers
  • the multicompartment delivery systems disclosed herein can have more than two distinct compartments ( Figures 2b and 2c).
  • the multicompartment delivery system can comprise 3 compartments.
  • the nanoparticles are solid and hydrophilic.
  • the nanoparticles can be encapsulated in a hydrophobic microsphere, which forms an outer hydrophobic compartment.
  • the hydrophobic microsphere can be composed of oil, such as safflower oil or any other biologically compatible oil, a wax, or hydrophobic molecules such as poly(epsilon-caprolactone), (PCL), poly(D,L-lactide-co-glycolide) (PLGA), and
  • microspheres can be obtained commercially from Spherotech, Inc. (Lake Forest, IL). Methods for making microspheres are also known in the art (see, e.g., Barbato et al. Biomaterials (2001) 22(11): 1371-8; Luciani et al. Biomaterials (2008) 29(36): 4800-7).
  • This structure can have a third compartment that encapsulates the microsphere. This compartment is an compartment ( Figures 2b and 2c).
  • the microsphere can be surrounded by a gel such as gelatin.
  • the microsphere can be surrounded by water.
  • the multicompartment delivery systems of the present disclosure can also be modified by target- specific binding agents that target the delivery systems to a cell or tissue.
  • target-specific binding agents are antibodies, Fab fragments and other antibody binding fragments, ligands, peptides, small molecules, and peptidomimetic compounds.
  • the target-specific binding agent is Tuftsin.
  • the target-specific binding agent is arginine-glycine-aspartate ("RGD") tripeptide.
  • the multicompartment delivery systems are targeted to macrophages using specific peptides functionalized on the surface of the outer compartment. For instance, the outer compartment of the multicompartmental delivery system can have target-specific binding agents attached to its surface.
  • the multicompartment delivery systems encapsulate therapeutic or imaging agents.
  • agents can be encapsulated within the inner compartment, outer compartment, or both.
  • the agent can promote pro- or anti-inflammatory properties (e.g., ovalbumin for pro-inflammation and immune-adjuvant response or omega-3 fatty acid-containing oils for anti-inflammatory response) to further enhance the therapeutic efficiency of the system.
  • the agents can be therapeutic agents such as chemotherapeutics, radioisotopes, antibiotics, peptides, vaccines, small molecules, proteins, and antibody
  • therapeutics can be nucleic acids used for gene therapy.
  • therapeutic agents are DNA molecules.
  • Agents can be siR A molecules delivered to silence genes in specific tissues.
  • One of the many advantages of the multicompartmental delivery systems disclosed herein is they utilize a compartmentalized delivery system which can be used to shield payloads prone to degradation until they have reached the target of interest. This is important for successful gene therapy, as macrophages, a preferred target cell, contain large amounts of nucleases within their lysosomes, making it likely that without the protection of these disclosed systems, that the DNA payload delivered to a cell would be degraded. . 2. Methods of Using Multicompartment Delivery Systems
  • the method of imaging and treating a tissue with a multicompartment delivery system disclosed herein comprises (a) administering an effective amount of the multicompartment delivery system to a subject, the delivery system comprising one or more inner compartments encapsulated within an outer compartment, the one or more inner compartments being about 10 nm to about 300 nm and the outer compartment being from out 0.5 microns to about 20 microns, the one or more inner compartments further comprising a first material having a first physical property, the outer compartment comprising a second material having a second physical property that is different from the first physical property, the one or more inner compartments encapsulates a first agent; (b) allowing the multicompartment delivery system to selectively bind to a target tissue or a cell; and (c) delivery of therapeutic agent, (d) detecting the imaging agent in the subject.
  • the inner compartment is in the size range of about 10 nm to about 30 nm.
  • the first agent is an imaging agent.
  • the first agent is colloidal gold, iron-oxide crystals, quantum dots, fluorophores, radiolabels, X-ray contrast agents, and positron emission tomographic agents.
  • the first agent is a therapeutic agent.
  • the first agent is selected from the group consisting of chemotherapeutics, antibiotics, antivirals, small molecules, siR A, and DNA.
  • the outer compartment encapsulates a second agent.
  • the second agent is an imaging agent.
  • This imaging agent can be selected from the group consisting of fluorophores, radiolabels, X-ray contrast agents, and positron emission tomographic agents.
  • the second agent is a therapeutic agent.
  • the outer compartment or inner compartment of the delivery vehicle can encapsulate the one or more imaging agents.
  • the term "encapsulate" means to enclose within or associate with the polymeric carrier. The encapsulation can include association of a molecule or compound at the surface of the polymeric carrier or within the polymeric carrier.
  • imaging agents include fluorophores, radiolabels, X-ray contrast, RAMAN spectroscopy agents, and positron emission tomography agents.
  • fluorophores include rhodamine, fluorescein, isothiocyanate, phycoerythrin, phycocyanin, fluorescamine, and metal chelates.
  • radiolabels include 3 H, 123 1, 125 1, 131 1, 32 P, 35 S, 14 C, 51 Cr, 36 C1, 57 Co, 55 Fe, 59 Fe, 90 Y, "mTc (metastable isomer of Technetium 99), and 75 Se.
  • X-ray contrast agents include Diatrizoate (Hypaque 50), Metrizoate (Isopaque 370), Ioxaglate (Hexabrix), Iopamidol (Isovue 370), Iohexol (Omnipaque 350), Ioxilan (Oxilan 350), Iopromide (Ultravist 370), and Iodixanol (Visipaque 320).
  • Other agents include 2,2,6,6- tetramethylpiperidine-l-oxyl (TEMPO) group, DID fluorescent dye analogs, and perdeuterated alkyl or aryl phosphonium groups.
  • the second agent is selected from the group consisting of chemotherapeutics, antibiotics, antivirals, small molecules, anti-inflammatory agents, siRNA, and DNA.
  • the first material is poly(epsilon-caprolactone). In other embodiments, the first material is hydrophilic. In still other embodiments, the first material is aqueous. In still others, the second material is hydrophobic. In some aspects, the second material is safflower oil.
  • the outer compartment is encapsulated within an exterior compartment.
  • the exterior compartment comprises an aqueous material.
  • a target-specific binding agent is attached to the surface of the outer compartment.
  • the target-specific binding agent is selected from the group consisting of antibodies and binding fragments thereof, peptide sequences, and ligands and binding fragments thereof.
  • the target-specific binding agent is an EGFR- specific antibody.
  • the target-specific binding agents bind to alpha-V-beta III integrin receptor.
  • a target-specific binding agent can be a small molecule that binds a target, ligand, peptide (e.g., arginine-glycine-aspartate ("RGD") tripeptide), protein, antibody, Fab fragment, F(ab')2 fragments, and peptidomimetic compounds.
  • peptide e.g., arginine-glycine-aspartate ("RGD") tripeptide
  • RGD arginine-glycine-aspartate
  • Exemplary targets include receptors (e.g., alpha- V-beta III integrin receptor and EGFR receptor), phospholipids, glycoproteins, and proteins.
  • the instant disclosure also teaches methods of treating a disease with a
  • the method comprising: (a) administering an effective amount of the multicompartment delivery system to a subject, the delivery system comprising one or more inner compartments encapsulated within an outer compartment, the one or more inner compartments comprising a first material having a first physical property, the outer compartment comprising a second material having a second physical property that is different from the first physical property, the one or more inner compartments being about 10 nm to about 300 nm and the outer compartment being from out 0.5 microns to about 20 microns, the one or more inner compartments further comprising a first therapeutic agent; (b) allowing the multicompartment delivery system to selectively bind to a target tissue or cell; and (c) releasing the therapeutic agent.
  • the disease is selected from the group consisting of atherosclerosis, Crohn's disease, inflammatory bowel disease, rheumatoid arthritis and cancer.
  • the one or more inner compartment is in the size range of about 10 nm to about 30 nm.
  • the one or more inner compartments or the outer compartments encapsulate an imaging agent.
  • the imaging agent is selected from the group consisting of colloidal gold, iron-oxide crystals, quantum dots, fluorophores, radiolabels, X-ray contrast agents, and positron emission tomographic agents.
  • the therapeutic agent is selected from the group consisting of chemotherapeutics, anti-inflammatories, fatty acids, antibiotics, antivirals, small molecules, siRNA, and DNA.
  • the method comprises a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutics, anti-inflammatories, fatty acids, antibiotics, antivirals, small molecules, siRNA, and DNA.
  • the second therapeutic agent is encapsulated by the outer compartment. In still other aspects, the second therapeutic agent is encapsulated by the inner compartment.
  • the first material is poly(epsilon- caprolactone). In still other embodiments, the first material is hydrophilic. In some aspects, the first material is aqueous. In still other embodiments, the second material is hydrophobic. In some aspects, the second material is safflower oil.
  • the outer compartment is encapsulated within an exterior compartment.
  • the exterior compartment can comprise an aqueous material.
  • a target-specific binding agent is attached to the surface of the outer compartment.
  • the target-specific binding agent is selected from the group consisting of antibodies and binding fragments thereof and peptide sequences.
  • the target-specific binding agent is an EGFR-specific antibody.
  • the target- specific binding agents bind to alpha- V-beta III integrin receptor.
  • the first agent is a gene construct.
  • the methods further comprise expressing the gene construct in the target tissue or cell.
  • the gene construct encodes IL-10.
  • the gene construct causes apoptosis in the target tissue or cells.
  • the gene construct is siRNA.
  • the target cell is a macrophage.
  • Interleukin-10 (IL-lO)-expressing plasmid DNA were formulated with gelatin nanoparticles that were encapsulated in a PCL microsphere.
  • the same delivery vehicle was used to orally administer systems disclosed herein to silencing the expression of tumor necrosis factor-a (TNF-a) in the treatment of inflammatory bowel disease.
  • An illustration of NiMOS and scanning electron micrographs of siRNA encapsulating gelatin nanoparticles and microspheres is shown in Figure 3A.
  • spherical gelatin nanoparticles, encapsulating siRNA with a mean diameter of 279.2 ⁇ 3.2 nm were produced (see, e.g., Oster et al.
  • nanoparticles were further encapsulated in PCL microspheres to create NiMOS. These were of fairly uniform shape and size with a relatively smooth surface morphology, and diameters ranging from 2.4 ⁇ to 3.2 ⁇ for blank and siRNA encapsulating microparticles, respectively ( Figure 3C).
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (The Dose Lethal To 50% Of The Population) And The ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds that exhibit large therapeutic induces are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the route of administration utilized for any compound used in the methods described herein, the
  • therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the disclosed systems and methods may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • physiologically acceptable carriers or excipients may be formulated for administration by, for example, injection, inhalation or insulation (either through the mouth or the nose) or oral, buccal, parenteral or rectal
  • the compounds can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade
  • the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g. , pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g.,
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Embodiments of the multicompartment systems disclosed herein can be designed for oral delivery to a subject.
  • the multicompartment delivery systems can comprise solid alginate and/or gelatin nanoparticles.
  • the hydrophilic nanoparticles can be encapsulated in a microsphere comprising a hydrophobic material such as poly(epsilon-caprolactone) (PCL) or poly(D,L-lactide-co-glycolide) (PLGA).
  • Figure 3 shows scanning electron microscopy images of these multi-compartmental delivery systems.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. , gelatin for use in an inhaler or insufflator may
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable delivery systems include microspheres which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology utilizes microspheres of precapillary size which can be injected via a coronary catheter into any selected part of the e.g. heart or other organs without causing inflammation or ischemia. The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells (e.g. endothelial cells).
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • oligomers can be formulated into ointments, salves, gels, or creams as generally known in the art.
  • a wash solution can be used locally to treat an injury or inflammation to accelerate healing.
  • the multicompartment delivery systems in the instant disclosure can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • the multicompartment delivery systems can be introduced systemically, e.g., by intravenous injection.
  • nucleic acid-encapsulated solid nanoparticles were designed that are further encased in the innermost water phase of the water-in-oil-in-water (W/O/W) multiple emulsion to develop nanoparticles-in-emulsion (NiE) multi-compartmental delivery system.
  • macrophages behave as a Trojan horse vector comes from the knowledge that micro-organisms, such as bacteria and viruses, use monocytes and macrophages to proliferate and hide from immune-surveillance system. Particularly in the case of HIV- 1 progression, there is mounting evidence depicting the role of macrophages in disease
  • HIV-1 has the ability to infect and replicate in non-diving cells such as cells of monocyte/macrophage lineage; although CD4+ T-lymphocytes appear to be the primary target of HIV virus.
  • the virus may affect the macrophages by influencing the cytokine production profile and this may result in chronic inflammation and extensive tissue damage.
  • the infected macrophages appear to be resistant to the toxic effects of the virus and thus serve as a Trojan horse to the HIV virus.
  • multicompartmental delivery systems can be utilized for gene therapy, gene silencing, cancer treatments, and vaccination.
  • the systems and methods described herein can be targeted to any tissue or cell type.
  • One such type of cell are macrophages. Nanoparticle Systems for Macrophage-Targeted Delivery
  • the over or under-activity of macrophages can result in the onset and/or progression of pathologic conditions, including, but not limited to, growth and spread of malignant tumors, sepsis, chronic inflammation in rheumatoid arthritis, lysosomal storage disease, atherosclerosis and major infections including HIV/ AIDS and tuberculosis.
  • Passive targeting refers to the accumulation of the drug or drug-carrier nanosystem by exploiting the pathophysiological condition and anatomical route.
  • nanoparticle systems that are >100 nm are readily opsonized and removed from the circulation by reticuloendothelial system (RES) and degraded by the macrophages in the liver and spleen.
  • RES reticuloendothelial system
  • the tendency of unmodified nanoparticles to localize in the RES has been exploited as a way to passively deliver the pay load to macrophages.
  • Nanoparticles have been observed to passively accumulate at inflamed sites due to the enhanced permeation and retention (EPR) effect. The presence of macrophages at these sites presents an opportunity to passively target these cells.
  • EPR enhanced permeation and retention
  • Active targeting approaches are based on the surface modification of the systems with an agent (e.g., ligand, antibody, peptide) that has the selective affinity for recognizing and interacting with the particular cell type, tissue, or organ in the body.
  • agent e.g., ligand, antibody, peptide
  • the phenomenon of active targeting can be achieved by utilizing the presence of various receptors and lipid components on the plasma membrane of cells. Macrophages are a preferred cell type for the systems disclosed herein. These receptors may be uniquely expressed on specific cells or may show a differentially higher expression on diseased cells in comparison to the normal cells. Table 1 below briefly describes some of the key properties of different receptors that are present on the surface of macrophages and can be utilized for actively targeting these cells. These different receptors can be downregulated or upregulated using known gene therapy techniques.
  • receptors can be used as targets for multicompartment delivery systems.
  • Such systems can have target-specific binding agents attached to the surface of the inner or outer compartment.
  • the target-specific binding agents can be ligands for a receptor, peptidomimetic compounds that bind to a receptor, or small molecules capable of binding to a receptor.
  • the multicompartment delivery systems are hydrophobic in certain embodiments. Since multiple emulsion globules are hydrophobic, they are readily internalized by intestinal macrophages upon oral administration through the Peyer's patch. Furthermore, when making these multicompartmental delivery systems, they can be generated in sizes that are preferentially taken up by macrophages. Once internalized by macrophages, these cells can travel and deliver the transfected protein in different parts of the body through systemic circulation. Alternatively, this method can also silence critical genes in activated macrophages and promote pro- or antiinflammatory status as desired for effective disease treatment of disease.
  • uptake by macrophages and efficient intracellular delivery also provides an opportunity for vaccination using peptides, proteins, attenuated microorganisms as antigens as well as DNA- and RNA-based vaccines that will require transfection and production of the appropriate human and cellular immune responses.
  • macrophages are known to phagocytose and kill various microorganisms, but with the advancement in the field of molecular biology, it has been established that many of these pathogens have developed subtle means for residing in the macrophages by mainly avoiding phagocytosis or developing lysosomal tolerance.
  • nanoparticle based drug delivery systems can improve the therapeutic index of such drugs by lowering the toxicity and enhancing the targeting ability to macrophages. See Table 2 for examples of nano-platform systems containing small molecule drugs for targeting macrophages.
  • Amphotericin B Visceral emulsions (nano Trilaurin/phosphatidylcholine 0.32 ⁇ 0.02 ⁇
  • macrophages that have taken up the multicompartmental delivery vehicles of the disclosed herein can be used to carry the payload delivered by said vehicle to a target site within the body, such as a tumor or site of inflammation.
  • the payload can be a therapeutic treatment, such as, but not limited to, anti-inflammatory therapeutics, including, but not limited to steroids, small molecule drugs (i.e. bisphosphonates), fatty acids, and antioxidants, as well as chemotherapy, antibiotics, peptides, vaccines, small molecules, proteins, and antibody therapeutics.
  • the present methodologies can also treat a range of anti-inflammatory diseases or conditions.
  • inflammatory disease or "inflammatory condition” as used herein is meant any disease, condition, trait, genotype or phenotype characterized by an inflammatory or allergic process as is known in the art, such as
  • the multicompartmental delivery systems disclosed herein are used to deliver a payload of a therapeutic treatment to a target cell, wherein the
  • Biological therapies such as therapeutic gene delivery or gene silencing can be designed to target and exploit macrophages.
  • the therapeutic gene can be transferred into the cells, which are then used as "guided missiles.”
  • Macrophages are capable of secreting various biological products (see Table 3 below). Depending on the products being secreted, if the process is left unchecked, this can result in tissue damage and fibrosis, which are characteristic of chronic inflammation. As the macrophage is capable of a number of processes in addition to secreting various bio-molecules, it is an ideal target for gene therapy for treatment of a variety of diseases and conditions, including, but not limited to, inflammation, cancer, Crohn's disease, inflammatory bowel disease, and atherosclerosis.
  • Reactive nitrogen intermediates e.g., NO
  • Neutral proteases elastase, lysozyme, acid hydrolases, collagenases, plasminogen activator, arginase, lipases, phosphatases
  • Coagulation factors Facts V, VIII, tissue factor
  • IL-1 IL-6, IL-10, IL-12, IL-15, IL-18, TNF- a, INF- a, TGF- ⁇ , GM-CSF, M-CSF,
  • Eicosanoids [0095]
  • the targets enumerated in Table 3 can be either up or downregulated depending on the desired result.
  • increasing expression of anti-inflammatory cytokines can be accomplished by delivering a gene construct to the cell using the multicompartmental delivery systems disclosed in the instant application. This would result in a reduction of the inflammatory response of the macrophages.
  • the antiinflammatory cytokine to be upregulated is IL-10.
  • siRNA can be administered to the cell using the multicompartmental delivery systems disclosed in the instant application.
  • the inflammatory cytokine to be downregulated or silenced is TNF-a.
  • Particles such as colloidal gold, iron-oxide crystals, and quantum dots are some of the examples of inorganic nanoparticles that are in the size range of 1- 30 nm. Methods of making such nanoparticles are known in the art (see, e.g.,, Luna Martinez et al. Mater. Sci. Forum: Adv. Elec. Microscop. Nanomat. (2010) 644: 51-55; Gardea-Torresdey et al. Journal ofNanoparticle Research (1999) 1(3): 397-404; Zhou et al. Chem. Mater. (1999) 11 (9): 2310-2312).
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • CT computed tomography
  • Macrophages are suitable imaging targets in diseases such as inflammation, atherosclerosis, and rheumatoid arthritis. This is because these cells are present in abundance and also are involved in multiple functions leading to inflammation and angiogenesis.
  • Nanoparticles designed to target macrophage cells have the potential to improve the detection and characterization of the human aortic atherosclerosis. Specific examples where the efficacy of the nanoparticles as contrast agents and targeting macrophages has been
  • Table 4 considers select examples of nano-platform systems, containing suitable imaging agents, that have been used to target macrophages and demonstrated the advantage of such systems in the treatment of atherosclerosis and rheumatoid arthritis disease models.
  • the classical activation state is characterized by killing of intracellular pathogens and tumor resistance and can be induced by interferon- ⁇ (IFN- ⁇ ) alone or in conjunction with microbial products such as lipo-polysaccharide (LPS) or cytokine such as tumor-necrosis factor alpha (TNF-a).
  • IFN- ⁇ interferon- ⁇
  • LPS lipo-polysaccharide
  • TNF-a tumor-necrosis factor alpha
  • Activation of macrophage via the classical pathway is marked by high antigen presentation capacity, high IL-12, IL-23, nitric oxide (NO), and reactive oxygen species production.
  • the alternative state can be induced by cytokines such as IL-4 and IL-13 and mainly results in anti-inflammatory responses and resolution of injury. Furthermore, the alternate activation stage is characterized by an increase in the IL-10 and IL-lra cytokines, mannose and scavenger receptors, arginase production and decrease in the production of inducible nitric oxide synthase enzyme. Figure 4 below highlights the central role played by macrophages in mediating innate as well as adaptive immune response.
  • the expression of inflammatory cytokines including but not limited to, tumor necrosis factor (TNF-a), can be down-regulated/silenced using the siR A (small interfering RNA) approach.
  • TNF-a tumor necrosis factor
  • Other embodiments of the systems and methods disclosed herein include treating inflammation by delivering plasmid gene encoding for anti-inflammatory cytokine protein such as IL-10 to macrophages, and comparing it to the silencing effect of TNF-a.
  • Table 5 describes some of the inflammatory mediators that are secreted by macrophages. Depending on the desired effect, the methods described herein can be used to modify the expression of any one of these mediators in order to control a disease state.
  • PDGF Platelet-derived growth factor
  • Proinflammatory cytokines IL-1, TNF-a, IL-6, IL-8
  • Chemokines MlPla, RANTES, ⁇ , MCP-1, MCP-3
  • IL-10 IL-12
  • IL-16 lymphocyte chemoattractant
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • IL interleukin
  • LTB 4 leukotriene B 4
  • LTC 4 leukotriene C 4
  • LTD 4 leukotriene D 4
  • MCP monocyte chemotatic protein
  • MIP macrophage inflammatory protein
  • PGD 2 prostaglandin D 2
  • PGE 2 prostaglandin E 2
  • PGF 2 prostaglandin F 2
  • RANTES regulated on activation normal T cell expressed and secreted
  • TNF- a tumor necrosis factor a.
  • IL-10 is produced by T cells, B cells, antigen presenting cells, mast cells and granulocytes amongst which macrophages are the major producers of IL-10.
  • IL-10 plays a significant role in immune regulation by its action on different cell types (See Table 6). When exposed to IL-10, monocytes and macrophages synthesize and release soluble immune mediators which bring about resolution of inflammation and help in tissue repair. These mediators include IL-IRA and soluble TNF-a receptor. Additionally, IL-10 inhibits the release of antiinflammatory cytokines including IL- ⁇ , IL-6, G-CSF, GM-CSF and TNFa.
  • Inflammation recruits macrophages that increase inflammation and can eventually cause tissue destruction.
  • the instant disclosure describes methods of modulating inflammation in order to stop the inflammatory cascade, enabling control and resolution of inflammation.
  • the anti-inflammatory cytokine IL-10 is delivered to a macrophage using the disclosed multicompartment delivery vehicle. Once within the macrophage, IL-10 is expressed by the cell, thereby reducing the inflammatory response caused by the macrophage.
  • siR A is delivered to a macrophage in order to stop the expression of inflammatory proteins secreted by the macrophage.
  • an IL-10 gene construct is administered utilizing the multicompartment delivery system disclosed herein. Once the multicompartment delivery system has been administered, it is allowed to selectively bind to a target tissue or a cell where it delivers its payload into the cell. Certain embodiments further comprise encapsulating the IL-10 gene construct in a nanoparticle. In some aspects the nanoparticle is designed to target an organelle within the cell. In certain aspects the organelle is the nucleus. In other embodiments, the multicompartment delivery system is comprises a second agent selected from the group consisting of a diagnostic, imaging, or therapeutic agent.
  • Gene therapy involves the delivery of genes to specific cells of the body where the gene encoding a protein is expressed. The resulting high concentrations of the desired protein should result in the treatment or prevention of a disease. Initially gene therapy was restricted only to genetic diseases where an abnormal or missing gene was delivered to replenish function of the missing gene. However, gene therapy can also be used for treatment of non-genetic diseases by supplementing genes encoding proteins capable of modulating cellular function.
  • compositions and methods disclosed herein can be used to perform gene therapy on diseased tissues and cells.
  • gene or “target gene” is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide.
  • a gene or target gene can also encode a functional RNA (fRNA) or non- coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
  • fRNA small temporal RNA
  • miRNA micro RNA
  • snRNA small nuclear RNA
  • siRNA small interfering RNA
  • snRNA small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • the cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus.
  • plants include monocots, dicots, or gymnosperms.
  • animals include vertebrates or invertebrates.
  • fungi include molds or yeasts.
  • nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, no more than 80 nucleotides in length, and no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
  • Oligonucleotides e.g., certain modified oligonucleotides or portions of
  • oligonucleotides lacking ribonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are
  • oligonucleotides make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.mol scale protocol with a 2.5 min coupling step for 2'-0-methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro nucleotides.
  • syntheses at the 0.2 .mu.mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I. sub.2, 49 mM pyridine, 9%> water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
  • R A including certain siNA molecules of the invention follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; and Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al, 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • common nucleic acid protecting and coupling groups such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.mol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-0- methylated nucleotides.
  • syntheses at the 0.2 .mu.mol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.
  • a 66-fold excess (120 .mu.L of 0.11 M 13.2 .mu.mol) of alkylsilyl (ribo) protected
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I. sub.2, 49 mM pyridine, 9% water in THF (PerSeptive
  • cytokine targeted protein and gene therapies have been explored in recent past. Antagonists of pro-inflammatory cytokines such as soluble TNF receptor molecule, IFN- yR-IgG fusion molecules and IL-12 receptor antagonist have been found useful in normalizing inflammatory response. Protein therapies, such as soluble TNF-receptor (etanercept), anti-TNF antibody (infliximab), IL-1 receptor antagonist (IL-IRa), were found to be efficacious but these required repeated high dose injections/infusions because of very short protein half life. Another limitation of protein therapy is due to the high systemic concentration of the protein therapeutic after injection. This may lead to undesired side effects of the therapeutic protein molecule at sites other than the target site in the body. These limitations can be overcome if genes encoding therapeutic protein are expressed in target cells leading to levels of protein which are high in the target tissue when compared to levels of protein in systemic circulation.
  • the present disclosed multicompartment delivery systems improve specificity of gene therapy approaches. Due to the higher specificity of targeted gene therapy, the methods disclosed herein lead to reduced systemic side effects. Furthermore, repeated administration of therapeutic proteins is not necessary when gene therapy is used since a single copy of plasmid DNA delivered inside a cell can undergo multiple expression cycles over a prolonged period of time. This allows for reduced dosing frequency.
  • Effective gene delivery systems would protect DNA from degradation and prevent their clearance by reticular endothelial systems. Furthermore, the gene delivery system should release its payload into the cytosol by endosomal escape, should facilitate nuclear entry of DNA and bring about high levels of gene transfection for a prolonged period of time.
  • the multicompartment systems disclosed herein can comprise polymers.
  • the polymers can be chemically modified.
  • chemical modification of cationic polymers can be carried out.
  • aggregation of positively charged particles can be reduced by increasing aqueous solubility of poly(L-lysine). This can be achieved by attachment of dextran to the epsilon amino groups of poly(L-lysine).
  • aqueous solubility can be increased by covalent coupling of PEG with amino groups of PEI.
  • modification of poly(L- lysine) with PEG can reduce its cytotoxic effect on cells in culture.
  • non-condensing polymers are capable of encapsulating DNA by non-electrostatic interaction. These polymers posses either a neutral charge or a slight negative charge, which helps protect DNA from exogenous nucleases. Absence of positive charge prevents electrostatic interactions between polymer and negatively charged DNA, making DNA encapsulation more feasible. Also, since the DNA is encapsulated within polymeric matrix, repulsion between the DNA payload and the cell membrane which may prevent endocytosis is restricted. Moreover, absence of positive charge limits recognition of non- condensing polymeric delivery vehicle by the mononuclear phagocyte system and hence limits early clearance of the delivery vehicle. In addition, non-cationic charge on polymer also avoids possibility of activation of innate immune system by mononuclear phagocyte system causing acute inflammatory response.
  • DNA encapsulating polymers like PEG, poly(ethylene oxide) (PEO), poly(D,L- lactide-co-glicolide) (PLGA), poloxamer block copolymers, gelatin and cellulose derivatives have been studied previously and have shown noticeable gene transfection efficiency in various studies.
  • PEG and PEO bring about DNA encapsulation by means of hydrogen bonds between nucleotide bases and phosphate groups of DNA and molecules of polymeric matrix.
  • Poloxamers Pluronics ® which are PEO-based copolymers have also been used for DNA encapsulation.
  • Type B gelatin-based nanoparticle vector for gene transfection has shown efficient systemic and local delivery, intracellular trafficking, and expression in various local and systemic studies (see, e.g., Magadala et al. AAPSJ. (2008) 10(4): 565-576). Nanoparticles formulated from Type B gelatin have several advantages like biocompatibility, biodegradability, high DNA loading capacity, high gene transfection efficiency, high cellular uptake by non specific or receptor mediated endocytosis.
  • Type B gelatin which is obtained by alkaline hydrolysis, has an isoelectric point between 4.8 and 5. At neutral pH Type B gelatin has net negative charge on its surface hence type B gelatin and DNA interact in a charge independent manner and DNA gets physically entrapped in hydrogel like matrix of gelatin biopolymer.
  • gelatin makes it a polymer of choice for its use as an efficient and safe non- viral gene transfection vector.
  • DNA can be provided with additional protection by encapsulation within nanoparticles, which can be then encapsulated in the innermost aqueous phase of a multiple emulsion.
  • a multi-compartmental DNA delivery vehicle can be formulated which can provide enhanced protection and controlled release property to encapsulated gene.
  • Multiple emulsion globules containing plasmid encapsulated nano-particles can release their payload either before its uptake by M cells of Peyer's patches in small intestine or after their engulfment by phagocytic cells present within Peyer's patches. Since multiple emulsion globules are hydrophobic, they are readily internalized by intestinal macrophages, as well as other cell types. Once internalized by macrophages, these cells can travel and deliver the protein in different parts of the body through systemic circulation.
  • the delivery systems of described herein involve the encapsulation of DNA within nanoparticles, allowing for the loading of a larger DNA payload in the emulsion system. This is because the DNA is protected within the nanoparticles and thus can withstand the high shear homogenization steps that occur during the process of preparing a multiple emulsion. Based on results obtained from gene transfection studies using gelatin nano-particles, it was believed that if these nanoparticles can be provided with further protection by their encapsulation in the innermost phase of water-in-oil-in-water (W/O/W) multiple emulsion system, controlled and enhanced gene delivery and transfection could be achieved both in vitro and in vivo.
  • W/O/W water-in-oil-in-water
  • Cancer is defined as the rapid and uncontrolled progression of cell growth. Tumor cells achieve this growth by undergoing an array of processes such as angiogenesis, metastases, and immunosuppression. Tumor-associated macrophages (TAM) seem to play a prominent role in tumor survival by helping the tumor to carry out abovementioned processes.
  • TAM Tumor-associated macrophages
  • TAMs are characterized by low expression of the differentiation-associated macrophage antigens, carboxypeptidase M and CD51 , high constitutive expression of interleukin (IL-1) and IL-6, and low-expression of TNF-a. It is believed that tumor-derived cytokines, chemokines, and proteases such as IL-4, 6, 10, TGF- ⁇ , and PGE2 act on macrophages so that they develop into polarized type II or M2 macrophages. As a result, unlike in healthy tissue, the anti-tumor activity of the macrophages is compromised in the tumor micro-environment.
  • the systems and methods disclosed herein provide for targeting macrophages. This allows the administration of therapeutics, such as chemotherapy or radioactive molecules for localized radiation therapy. Furthermore, the instant disclosure enables one of skill in the art to administer gene therapy using the
  • Gene constructs can be used to retrain TAMs to attack tumor cells, up or downregulate various biomolecule secretions on the part of the macrophage, as well as to delivery payloads to a tumor site.
  • Nanoparticle distribution within the body is based on various parameters, such as their relatively small size resulting in longer circulation times and their ability to take advantage of tumor characteristics.
  • W/O/W multiple emulsions are triphasic systems composed of three phases, inner aqueous droplets contained in oil globules surrounded by outer aqueous phase.
  • the W/O/W multiple emulsion was formulated by a two step emulsification.
  • Safflower oil (Jedwards, Inc., Quincy, MA), which is rich in omega-3 and omega-6 polyunsaturated fatty acids, was used for the oil phase of the emulsion.
  • Safflower oil contains high levels of tocopherols, which are reported to have a negative effect on levels of pro-inflammatory cytokines tumor necrosis factor alpha and interleukin 1-beta.
  • Tocopherols have shown to exert antioxidant activity and may provide protection against acute inflammation mediated by reactive oxygen species (10).
  • the first step consisted of formulation of W/O primary emulsion using an oil soluble surfactant Span ® 80. Naked plasmid (ME) or plasmid encapsulated in the gelatin nanoparticles (NiE) was incorporated in the aqueous phase of the primary emulsion to give the final concentration of 50 ⁇ g/ml of the multiple emulsion.
  • a stable primary W/O emulsion with encapsulated naked plasmid DNA or as dispersion with DNA-encapsulated nanoparticles was formed using the Silverson homogenizer L4RT at the speed of 9,000 rpm for 15 minutes.
  • the primary emulsion of dispersion was mixed with additional aqueous phase consisting of a water soluble surfactant Tween ® 80 and the formulation was prepared by homogenization at 4,000 rpm for 4 minutes. Increasing the speed to greater than 4,000 rpm during this step resulted in rupture of the multiple emulsion globules.
  • the cells were washed with sterile phosphate buffered saline (PBS, pH 7.4) and the cover slip was removed from the wells and mounted on a clean glass slide.
  • PBS sterile phosphate buffered saline
  • the microscopy images in Figure 1 (top) shows that that the rhodamine-dextran containing W/O/W emulsion and NiE formulations were rapidly internalized in murine alveolar macrophages J774A.1 cells. As time progressed, an increase in fluorescence intensity was observed, which became maximum after 120 minutes of exposure.
  • the microscopy images in Figure 1 (bottom) shows that maximum gene transfection was observed in cells treated with NiE formulations followed by ME and GNP formulations. Maximum green fluorescence was observed in cells treated with NiE formulation starting 24 hour post treatment with sustained levels at day 2, 3 and 4 followed by a decline at day 5 and 6.
  • NiE formulation was better compared to ME or GNP formulations in causing gene transfection since NiE formulation offer more protection to plasmid DNA during formulation resulting in higher loading efficiency of plasmid DNA per ml of formulation as well as during intracellular transport of plasmid DNA in endosomal/lysozomal compartments.
  • Macrophage-targeted tuftsin-modified cross-linked alginate nanoparticles were developed for local and systemic anti-inflammatory gene therapy. Plasmid DNA expressing both enhanced green fluorescent protein (GFP) and interleukin-10 (IL-10) were physically encapsulated in the non-condensing alginate matrix. Alginate nanoparticles could efficiently encapsulate the plasmid and protect against DNAse degradation. Furthermore, the tuftsin- modified nanoparticles were efficiently internalized in J774A. 1 macrophage cells with saturation within 1 hour of incubation.
  • GFP enhanced green fluorescent protein
  • IL-10 interleukin-10
  • IL-10 IL-10-expressing plasmid DNA and transfection for local and systemic antiinflammatory therapy.
  • the clinical significance of IL-10 applies in inflammation related diseases including, but not limited to, inflammatory bowel disease, atherosclerosis, and rheumatoid arthritis.
  • the expression of IL-10 in macrophages would result in termination or at least a significant reduction of the inflammatory response associated in such disease models.
  • an alginate carrier was selected as a biocompatible non-condensing polymer for preparation of nanoparticles and the surface was modified with tuftsin for macrophage-selective uptake.
  • GFP- or IL-10-expressing plasmid DNA 400 ⁇ g was mixed with an aqueous solution of medium viscosity alginate polymer (0.1% w/v) and stirred for an hour.
  • the plasmid DNA-containing alginate solution was then cross-linked by drop-wise addition of calcium chloride (9 mM).
  • the particles were then surface modified with 1 mg/ml of tuftsin peptide solution to target specific receptors on macrophages ( Figure 6).
  • the control and peptide- modified nanoparticle formulations were characterized for particle size, surface charge, surface morphology, and DNA loading efficiency. Stability of encapsulated DNA was assessed by agarose gel electrophoresis.
  • Toxicity of particles was evaluated by MTS assay in J774A.1 macrophage cell line. Time dependent cell uptake studies were carried out by encapsulating rhodamine-labeled dextran dye in nanoparticles and accessed via flow cytometry and
  • the particle characterization studies showed that the DNA-loaded cross-linked alginate nanoparticles formed were spherical in shape with a mean diameter of approximately 300 nm - 400 nm. On average, the surface charge of the unmodified formulation and the peptide-modified formulation were -47.7 mV and +19.7 mV, respectively, which also indicated successful peptide conjugation on the surface of the nanoparticles. Both GFP- and IL-10 expressing plasmid DNA were optimally encapsulated with an efficiency of approximately 58% and the stability studies confirmed that DNA remained intact during the formulation process.
  • Results of this study show that NiE formulations were more efficient in transfection of EGFP-N1 in alveolar macrophages as compared to the control formulations such as DNA dissolved in the aqueous phase of the W/O/W multiple emulsion (ME), DNA encapsulated in GNP, or when DNA was complexed with Lipofectin®, a cationic lipid-based transfection reagent.
  • ME W/O/W multiple emulsion
  • Lipofectin® a cationic lipid-based transfection reagent.
  • Gene therapy involves delivery of therapeutic genes into specific cells followed by transcription and translation into biologically-active protein for treatment of different acute and chronic diseased conditions.
  • gene sequences can be used for production of desired protein within specific cells in the body (1).
  • development of a delivery vehicle that is able to efficiently deliver genes into specific cells is useful.
  • viral vectors have high gene transfection efficiency, their clinical use has been limited due to cytotoxicity, immunogenicity, and chromosomal integration of delivered genes.
  • Non-viral gene delivery systems targeted towards macrophages provide a unique way to control acute and chronic inflammatory diseases.
  • NiE was developed as a novel macrophage-targeted system for gene delivery and transfection, especially following mucosal administration.
  • Such a multi-compartmental delivery system also provides a way to encapsulate different types of therapeutic or imaging payloads in the inner and outer
  • the disclosure provides results obtained from in vitro gene transfection experiments using EGFP-N1 plasmid DNA in murine adherent alveolar macrophages J774.A1 cells.
  • Type B GNP was prepared by ethanol-water solvent displacement method as described previously (3). Particle size and charge were determined using Malvern Zetasizer®. W/O/W multiple emulsion was formulated by a two step emulsification process using a
  • Silverson homogenizer® L4RT (5) For cellular uptake studies, rhodamine-labeled dextran, a water-soluble fluorophore, was incorporated in the internal aqueous phase of ME or encapsulated in GNP, which were then incorporated in the inner aqueous phase. Cellular uptake studies were performed by fluorescence microscopic imaging of control and treated J77A.1 cells. For gene transfection studies, EGFP-Nl was encapsulated in GNP, which was then incorporated in the inner aqueous phase of the ME. EGFP-Nl loading in different formulations was evaluated using PicoGreen® dsDNA reagent.
  • EGFP-Nl stability in different formulations was characterized by using agarose gel electrophoresis.
  • Cells treated with different EGFP-Nl encapsulated formulations and complexed with Lipofectin® was examined for transgene expression qualitatively by fluorescent microscopy and quantitatively by green fluorescent protein (GFP)- specific enzyme-linked immunosorbent assay (ELISA).
  • GFP green fluorescent protein
  • the blank and EGFP-Nl -loaded GNP had an average particle size of 164.3 nm and 166.0 nm, respectively.
  • the surface charge of blank and EGFP-Nl -loaded gelatin nanoparticles was found to be - 10.7 mV and -11.2 mV, respectively.
  • ME and NiE had globule size of 5 ⁇ or less with no signs of leakage of contents from the inner aqueous phase ( Figure 10).
  • Loading efficiency of EGFP-Nl in GNP, ME, and NiE was found to be 99%, 54%, and 70%,
  • GFP green fluorescent protein
  • This example evaluated down-regulation of TNF-a by oral RNA interference therapy.
  • Control scrmbled sequence
  • siRNA TNF-a specific small interfering RNA
  • mice Upon confirmation of the dextran sulfate sodium (DSS)-induced acute colitis model, mice were divided into several treatment groups receiving no treatment, blank NiMOS, NiMOS with scramble siRNA, or NiMOS with TNF-a silencing siRNA by oral administration.
  • DSS dextran sulfate sodium
  • IBD Inflammatory bowel disease
  • Crohn's disease and ulcerative colitis - is a chronic relapsing condition involving inflammation of the gastrointestinal tract. While its etiology is still unknown, a complex interaction of genetic disposition, environmental conditions, life style, microbial and immune factors have been identified as some of the major aspects leading to IBD (1, 2).
  • Conventional treatment consists of anti-inflammatory and immune-suppressive drugs (3), depending on the patient's clinical state including extent and severity of the disease. Some medications are effective measures to combat inflammation in the acute setting, but ineffective in maintaining remission due to toxicity, dependency and higher relapse rates.
  • Tumor necrosis factor-alpha plays a central role in the mediation of inflammation in IBD (5). While systemic anti-TNF-a works well in patients refractory to conventional therapy, serious contraindications and side effects including opportunistic infections, antibody formation against anti-TNF-a in conjunction with decreased efficacy of the therapy, and increased occurrence of infusion reactions have been reported (6, 8, 9).
  • the gastrointestinal tract offers an ideal target for gene therapy due to the large surface area and access to the luminal site of inflammation via oral and rectal routes of administration.
  • RNA interference therapy utilizes short interfering RNA (siRNA), usually composed of 20-25 nucleotides, and is involved in a gene-silencing mechanism through RNA interference where siRNA can ultimately block the expression of a specific gene, e.g. those expressed in diseases.
  • siRNA short interfering RNA
  • encapsulation and delivery mechanisms have been employed involving various systems including viral vectors, nanoparticles 18, polymer-based vehicles or liposomal vesicles.
  • NiMOS multicompartmental nanoparticles-in- microsphere system
  • the control was encapsulated in gelatin nanoparticles (forming NiMOS).
  • TNF-a specific siRNA was also encapsulated in gelatin nanoparticles. This was done in order to evaluate the potential of oral delivery for TNF-a gene silencing in IBD by administration in a DSS-induced acute colitis mouse model.
  • Type-B gelatin (MW 40,000-50,000, 100-115 mM of free carboxylic acid per lOOg of protein, pi of 4.7-5.2) of 225 bloom strength and Poly(vinyl alcohol) (PVA) (degree of hydrolysis 86.7-88.7%, MW -67,000) were obtained from Sigma Chemical Company, (St.
  • PCL Poly(epsilon-caprolactone) (MW 10,000-20,000 as verified by gel permeation chromatography) was purchased from Polysciences, Inc. (Warrington, PA). Reagent grade dichloromethane, haematoxylin and eosin were purchased from Fisher Scientific
  • siRNAs were obtained from Dharmacon (Lafayette, CO). The sequences were as follows: TNF-a siRNA, 5 'GACAACCAACUAGUGGUGCdTdT3 '; scramble siRNA, 5 'GACAACCAGGGCGUGGUGCdTdT3 ' 14. Only sense sequence is shown.
  • DSS dextran sodium sulfate
  • RNAse A and Superscript III were obtained from Invitrogen (Carlsbad, CA).
  • Gelatin nanoparticles were prepared as described previously. In brief, siRNAs were mixed with aqueous gelatin solutions of pH 7.00 and pre-incubated for 10 min at room temperature followed by controlled precipitation of siRNA-containing or blank gelatin solutions with the aid of ethanol as the non solvent. In the final blend, the ethanol-to-water volume ratio was kept constant at 65:35. The resulting nanoparticles were centrifuged at 32,000rpm for 45 min, washed with deionized water and lyophilized.
  • Nanoparticle-in-microspheres-oral delivery systems were manufactured by utilizing a "double emulsion-like" technique, as described herein.
  • siRNA- loaded gelatin nanoparticles and NiMOS were prepared as discussed above. Encapsulation efficiency of siRNA in these formulations was determined by dissolving a known amount of sample in phosphate buffered saline (PBS, pH 7.4) containing 0.2 mg/ml protease at 37°C until a clear solution was obtained. Released siRNA was quantified using the PicoGreen® assay (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The intensity of fluorescence which is proportional to the PicoGreen® bound siRNA was measured at an excitation of 485nm and emission of 520 nm wavelengths using a Perkin Elmer LS50B spectrophotometer (Norwalk, CT). FLWINLAB® software was used for processing and analysis of resulting data.
  • PBS phosphate buffered saline
  • RNAse A A sample of siRNA containing NiMOS was treated with RNAse A for 20min at 37°C followed by inactivation of the enzyme and extraction of siRNA from loaded nanoparticles by protease digestion of the gelatin matrix that were extracted in the same manner as discussed above.
  • siRNA release from microspheres and nanoparticles samples were added to the wells of a 4% (w/v) pre-stained E-gelTM system and run against a lObp ladder, naked siRNA, naked siRNA treated with protease, and naked siRNA treated with protease and RNAse A as controls. After a 45 min run at 70V, the image was recorded under UV light and processed.
  • mice Female Balb/c mice (6-8 weeks, ⁇ 18-20g) were purchased from Charles River Laboratories (Wilmington, MA). Animals were randomly assigned to groups of 4 mice per cage and acclimatized for 10 days before begin of the study. The mice were housed in rooms at a controlled temperature of 22°C and 26% humidity, respectively, with light-dark cycles of 12 hours and fed with water and a standard pellet at libitum except when fasted overnight to prepare for the oral gavage.
  • DSS Dextran Sodium Sulfate
  • DSS dextran sodium sulfate
  • mice of each group including all controls were fasted overnight followed by treatment with blank NiMOS (particles containing no siRNA) or microparticles containing 750pmol siRNA (TNF a or TNFa scrambled sequence) per animal by oral gavage using a blunt-tipped feeding needle inserted into the esophagus adding up to three doses of NiMOS formulations per group.
  • An additional control group consisted of animals receiving no oral treatment throughout the course of DSS exposure (DSS control).
  • RNAlater® Applied Biosystems Inc., Foster City, CA
  • RNAqueous® - 4PCR kit (Applied Biosystems Inc., Foster City, CA) according to the
  • RNA from the tissue samples described above was quantified by using the NanoDrop 1000 (Thermo Fisher Scientific Inc., Wilmington, DE) UV- Vis spectrophotometer.
  • Synthesis SuperMix for qt-PCR (Invitrogen, Carlsbad, CA) as recommended by the manufacturer.
  • the preparation for reverse transcription of RNA samples was performed on ice and in accordance with the manufacturer's recommendation.
  • a volume corresponding to 1 ⁇ g of RNA as determined by UV spectrophotometric analysis was used for the synthesis process.
  • Samples containing cDNA were diluted to a final volume of 100 ⁇ and stored at -20 °C until further use.
  • Quantitative real time PCR was performed on cDNA samples obtained from large intestinal tissue to determine the levels of mRNA transcript.
  • L32 gene expressing the L32 ribosomal protein served as a control.
  • Pre-diluted cDNA was mixed with 0.2 ⁇ of primer pair detecting the murine TNF-a or L32 construct and SYBR® Green PCR master mix and pipetted into an ABI PrismTM 96-well optical reaction plate with barcode (Applied Biosystems, Foster
  • samples of the entire large intestine were homogenized in lysis buffer (1 M Tris, pH 7.4; 0.5 M EDTA, pH 8.0; 5 M NaCl, 10% (w/v) Brij, 10% (v/v) Tween 20), supplemented with proteinase inhibitor, on ice to extract the proteins from the tissue samples. Protein containing supernatant was separated by centrifugation at 13000 g for 30 min at 4°C and stored at -70°C until analysis.
  • concentrations obtained from the multiplex ELISA were normalized against the total protein content of each individual sample as determined by bicinchoninic acid assay (Pierce, Rockford, IL). Values are expressed as pictogram (pg) or femtogram (fg) of murine cytokine expressed per mg of total protein content of each sample and represent mean ⁇ S.D. of 4 mice.
  • Tissue samples were evaluated for mucosal architectural change, cellular infiltration, inflammation, external muscle thickening, presence of crypt abscess, goblet cell depletion, signs of edema, surface epithelial cell hyperplasia, and signs of epithelial regeneration by using light microscopy of hematoxylin and eosin survey staining (25, 35). These values were used to assess the degree of mucosal damage and repair in treatment and control groups. Tissue samples harvested from distal regions of the non-/inflamed colon were stored in 10% formalin solution at 4°C for at least 2 days to fix the tissue.
  • Tissue sections with a thickness of 7 ⁇ were stained with haematoxylin and eosin according to protocols supplied by the manufacturer followed by imaging using bright-field microscopy (Olympus BX51TRF, Olympus America, Center Valley, PA). Determination of Tissue Myeloperoxidase Activity
  • Tissue myeloperoxidase activity was detected with the FluoroMPO kit purchased from Cell Technology, Inc. (Mountain View, CA).
  • FluoroMPO kit purchased from Cell Technology, Inc. (Mountain View, CA).
  • tissue samples were minced in hexadecyltrimethylammonium bromide buffer (0.5% in 50 mM phosphate buffer) on ice and homogenized. The homogenate was sonicated, subjected to a freeze- thaw cycle and centrifuged at 10,000 rpm for 3 minutes. A certain known amount of the tissue lysate
  • the detection reagent is oxidized in the presence of hydrogen peroxide and MPO to produce its fluorescent analog which was measured at an excitation wavelength of 530nm and emission at 590 nm. Reported values a normalized to mg of total protein content of the sample.
  • FIG. 13 A An illustration of NiMOS and scanning electron micrographs of siRNA encapsulating gelatin nanoparticles and microspheres is shown in Figure 13 A.
  • spherical gelatin nanoparticles encapsulating siRNA with a mean diameter of 279.2 nm ⁇ 3.2 nm were produced.
  • the nanoparticles were further encapsulated in PCL microspheres to create NiMOS. These were of fairly uniform shape and size with a relatively smooth surface morphology, and diameters ranging from 2.4 ⁇ to 3.2 ⁇ for blank and siRNA encapsulating microparticles, respectively (Figure 13C).
  • mice After 5 days of DSS administration in drinking water, mice showed typical symptoms of IBD including weight loss (Figure 14 A), diarrhea, rectal bleeding, and signs of inflammation as evidenced by elevated myeloperoxidase levels (Figure 14B), a significant degree of white blood cell infiltration, and abnormal mucosal architecture including goblet cell depletion (Figure 14C).
  • murine TNF-a protein levels were determined using a commercially available ELISA (Figure 15B). Overall, the group receiving TNF-a NiMOS had the lowest expression levels at both time points with an approximate three-fold decrease on day- 14. Protein concentration was the highest on day-10 and 14 in the DSS control group. Here, an approximate 5-fold increase was observed as compared to levels measured in TNF-a NiMOS treated animals.
  • Interleukin (IL)-la and IL- ⁇ ⁇ were up-regulated on day-10 in the DSS control group reaching values of 135 and 57 pg/mg total protein, respectively ( Figures 16 A, 16B). No significant decrease in concentration was observed on day-14. In all other NiMOS groups, less than half the expression levels were detected when compared with the blank NiMOS treatment group for IL-la on day-10. However, on day-14, concentrations of IL-la and IL-1 ⁇ were lowest in the TNF-a NiMOS group in contrast to all other colitis groups showing more than 5- and 3- fold down-regulation compared to the DSS control with no significant difference in remaining groups.
  • GMCSF granulocyte macrophage colony-stimulating factor
  • ⁇ - ⁇ monocyte inflammatory protein
  • Figure 16K shows that both chemokines were significantly decreased on day-14 in the TNF-a NiMOS group, reaching values of 0.45 pg GMCSF and 4.5 pg ⁇ - ⁇ per mg total protein for all colitis groups. This is equivalent to a 10-fold and more than a 6-fold decrease of the chemokines as compared to the colitis group and a 6-fold and 3-fold decrease compared to the blank and scramble NiMOS, respectively. Blank and scramble NiMOS exhibited similar levels of these two chemokines, with values of about 2.7 pg GMCSF and 13 pg ⁇ - ⁇ per mg total protein. A similar result was observed for monocyte chemotactic protein (MCP)-l for day-14.
  • MCP monocyte chemotactic protein
  • H&E stained cryosections from the large intestine were evaluated for changes occurring at the histological level upon induction of acute colitis and treatment of inflammation with TNF-a NiMOS (Figure 17).
  • Tissue from the control group had normal colon histology bearing no signs of inflammation or disruption of healthy tissue morphology, serving as the baseline for comparison to the remaining samples.
  • intestinal tissue from the DSS control, the group receiving blank NiMOS, and scramble NiMOS group exhibited clear signs of inflammation including cell infiltration, goblet cell depletion, and irregular mucosal structure as described before, which did not substantially subside as the study progressed.
  • tissue from the TNF-a NiMOS group showed a considerable decrease in the level of
  • mice Changes in body weight of colitis-induced mice were evaluated after administration of various treatments in comparison to the control ( Figure 18). On days 2, 4, and 6, mice from all groups were fasted overnight for consistency, as food in the stomach and other sections of the gastrointestinal tract is known to interfere with dosing and analysis upon administration of the formulations. The following morning they were orally administered with blank, TNF-a or scramble NiMOS. Since fasting caused a weight loss of approximately 10% in all groups (Figure 18 A), mice were administered every other day to allow for normalization of body weight to occur. The weight in the control group stabilized after the last administration leveling out at approximately 5% higher than the weight at the beginning of the study.
  • Weight loss for the DSS control, blank and scramble NiMOS animals was quick after the last oral administration with respective values of 19%, 26%, and 18% loss of original body weight on day-14.
  • the smallest weight loss of approximately 8% was observed in the TNF-a NiMOS group.
  • general appearance was much closer to a healthy mouse in this group with no severe symptoms of colitis as opposed to the remaining colitis mice described before, which exhibited the classical features associated with acute colitis in a considerable number of animals.
  • Colon length was assessed and is reported in Figure 14B. Control mice had a colon length of approximately 8.8 cm. A significant reduction in colon length was observed upon induction of DSS colitis with values of approximately 6 cm. Mice treated with blank and scramble sequence siR A NiMOS also exhibited shortening of the colon comparable to the colitis control. For the TNF-a NiMOS group, an increase in colon length to almost 8 cm was observed, which was closer to the baseline compared to the remaining colitis groups.
  • the instant disclosure uses local IBD treatment that combines positive aspects of RNA interference with the safety of biodegradable polymeric nano- and microparticulate systems enabling a localized treatment through oral administration of siRNA.
  • This option might offer several advantages including lower/fewer doses due to localized therapy and no relative dilution of the agent associated with systemic administration, fewer side effects and higher efficacy.
  • RNA silencing for TNF-a was successfully encapsulated, followed by oral administration to DSS-induced colitis mice and evaluated efficiency and feasibility of RNA silencing involved in inflammation.
  • the disease model was chosen because it is one of the most frequently used rodent IBD models. Supplying DSS in the drinking water of animals at libitum provides a predictable onset and course of the disease and lower risk of mortality.
  • This platform has been shown to encapsulate plasmid DNA efficiently, enable intracellular uptake, and nuclear transport while being safe and efficient for gene transfection; and it can easily be adapted for encapsulation of siRNA.
  • type B gelatin was selected for the nanoparticle formulation because it can physically entrap pDNA and siRNA, protecting the payload during intracellular transport; whereas other, more common systems rely on surface adsorption or electrostatic complexation to carry siRNA.
  • NiMOS can effectively protect the siRNA encapsulated within the nanoparticles inside PCL microspheres from the gastrointestinal barriers and degrading proteolytic enzymes; and release is expected to occur in the intestines at the site of inflammation.
  • the microspheres with sizes smaller than 5 ⁇ promote localization in the intestine by delivery of gelatin nanoparticles to the enterocytes and other cells at these sites once the PCL matrix is degraded by lipases abundantly present in the intestinal tract allowing for endocytosis. As such, this is an effective system for siR A encapsulation and delivery.
  • TNF-a NiMOS the analysis of real time PCR of both time points showed very low levels of murine TNF-a, which was similar to the level observed in healthy, control mice on day- 10. This is attributed to the silencing effect of NiMOS, which was not observed in remaining colitis-bearing groups.
  • the relative decrease in expression of TNF-a mRNA in all NiMOS groups on day-14 can potentially be attributed to a complex interplay of pro-and anti-inflammatory cytokines resulting in compensatory responses due to induction of colitis.
  • ELISA results were in good correlation with real time-PCR analysis.
  • TNF-a NiMOS TNF-a NiMOS animals
  • TNF-a siRNA NiMOS RNA silencing
  • proinflammatory cytokines where detected in blank and scramble NiMOS groups.
  • Lower expression of many cytokines due to down-regulation of TNF-a could result in alleviation of inflammation at the disease site. This was also reflected in the smaller degree of weight loss and in colon tissue morphology which showed signs of regeneration with only few abnormalities.
  • a gelatin, non- condensing polymer can bring about efficient gene transfection.
  • Evaluations of green fluorescent protein expressing plasmid DNA (EGFP-Nl) trans fection efficiency were carried out murine adherent alveolar macrophages J77A.1.
  • Gelatin nanoparticles were prepared by a solvent displacement method. Briefly, 100 mg of type B gelatin bloom strength 225 (Sigma Aldrich) was dissolved in 10 ml of water at 37°C. After this step, the pH was adjusted to 7 using 0.2 M NaOH solution. Gelatin particles were precipitated from the aqueous solution using ethanol as an anti-solvent. The final ratio of ethanol water was optimized to 4: 1. Prior to its addition to gelatin solution, the temperature of ethanol solution was brought down to -40°C in order to enhance the anti-solvent effect.
  • type B gelatin bloom strength 225 Sigma Aldrich
  • Enhanced green fluorescent protein (EGFP) expressing plasmid DNA (EGFP-Nl) was added at concentration of 1% w/w of solubilized gelatin.
  • rhodamine-conjugated dextran (Mol. wt. 70kDa) was added at final concentration of 0.5% w/w of gelatin to gelatin solution after pH adjustment for its physical encapsulation.
  • the precipitated particles were centrifuged at 35,000 rpm for 45 minutes using a Beckman ultracentrifuge.
  • the nanoparticle pellet was collected, washed with deionized water, flash frozen in liquid nitrogen, and lyophilized for 24 hours to give a dry powder.
  • Nanoparticles-in-Emulsion Formulation: Water-in-oil-in-water (W/O/W) multiple emulsions are triphasic systems composed of three phases, inner aqueous droplets contained in oil globules surrounded by outer aqueous phase. The W/O/W multiple emulsion was formulated by a two step emulsification method. Extra pure safflower oil
  • Safflower oil contains high levels of tocopherols, which are reported to have a negative effect on levels of pro-inflammatory cytokines tumor necrosis factor alpha and interleukin 1 beta. Also, tocopherols have been shown to exert antioxidant activity and may provide protection against acute inflammation mediated by reactive oxygen species.
  • the first step consisted of the formulation of W/O primary emulsion using an oil soluble surfactant, Span ® 80. Naked plasmid (ME) or plasmid encapsulated in the gelatin nanoparticles (NiE) was incorporated in the aqueous phase of the primary emulsion to a final concentration of 50 ⁇ g/ml of the W/O/W multiple emulsion. The appropriate amounts of each ingredient in the optimized formulations are listed in Table 7. [0199] A stable primary W/O emulsion with encapsulated naked plasmid DNA or as dispersion with DNA-encapsulated nanoparticles was formed using the Silverson homogenizer
  • Table 7 The Composition of the Optimized Primary and Multiple
  • Plasmid DNA Loading Studies Loading efficiency of pEGFP-Nl in NiE, ME and GNP formulations was evaluated by using Quant-iTTM PicoGreen® dsDNA Reagent (Invitrogen, Carlsbad, CA). PicoGreen® dsDNA Reagent is a highly sensitive fluorescent stain for quantification of double stranded DNA.
  • NiE and ME were loaded with pEGFP- Nl at final concentration of 50 ⁇ g/ml in the internal phase of multiple emulsion.
  • GNPs were loaded with l%w/w of pEGFP-Nl, 40 mg of which were incorporated in the internal phase of the multiple emulsion.
  • Extraction of encapsulated pEGFP-Nl from lyophilized GNP was carried out by incubating 1 mg of gelatin particles with 1 ml of PBS containing 0.2 mg/ml protease for 30 minutes at 37°C.
  • pEGFP-Nl was extracted by first destabilizing emulsion by addition of 1/10 volume of 5M NaCl followed by centrifugation at 20,000 rpm for 30 minutes.
  • the oil and water layers became separated in to two distinct layers.
  • the aqueous layer was diluted 200 times to concentration of 2 ⁇ g/ml with PBS. Ten ⁇ of this diluted solution was used for estimation of DNA loading.
  • NiE 1 ml of emulsion was de-stabilized with 100 ⁇ of 5M NaCl and centrifuged at 20,000 rpm for 30 minutes.
  • the GNP palette obtained was treated with 5ml of protease buffer for 30 minutes at 37°C for release of EGFP-N1 plasmid DNA from the particles.
  • Blank gelatin particles and emulsions with blank inner aqueous phase or blank gelatin particles in the inner aqueous phase were used as controls after same extraction treatment as plasmid loaded formulations was performed.
  • Stability of EGFP-N1 Plasmid DNA A gel retardation assay was performed to evaluate stability of plasmid DNA encapsulated in NiE, ME and GNP to ensure physical and chemical stability of plasmid DNA during the process of formulation.
  • E-Gel® 1.2% with SYBR SafeTM (Invitrogen, Carlsbad, CA) were used.
  • SYBR safe DNA stain has an advantage that it is safer compared to ethidium bromide which can cause mutations and chromosomal transformations.
  • Agarose gel electrophoresis involves movement of DNA through agarose gel under influence of electric field in which negatively charged DNA moves towards positive end through the gel.
  • EGFP-Nl plasmid DNA was extracted from NiE, ME and GNP formulations as mentioned above and loaded at concentration of lOOng in 20 ⁇ 1 of loading buffer per well of 1.2% E-Gel cassette® (Invitrogen, Carlsbad, CA).
  • EGFP-Nl plasmid DNA loading efficiency was calculated as percentage of plasmid loaded compared to plasmid added initially. Results shown in Table 9 show that plasmid loading efficiency was increased, when plasmid was protected by encapsulation in GNP in NiE formulation compared to ME formulation in which unprotected naked plasmid was present in the internal phase of ME. Later a separate group of blank emulsion formulations were terminally spiked with EGFP-Nl plasmid DNA after completion of both steps of emulsification process. Then similar plasmid extraction procedure was carried out. Amount of plasmid assayed from these experiments was found to be 99.16% ⁇ 0.35 of that added terminally. This result indicated that decrease in plasmid loading efficiency was might be due to high shear homogenization process.
  • Table 9 Plasmid DNA encapsulation efficiency in type B gelatin particles and in nanoparticles-in-emulsion formulations
  • DMEM fetal bovine serum
  • penicillin/streptomycin antibiotics were allowed to divide until they reached desired density. Cell count was estimated by placing 20 ⁇ of the cell suspension mixture on a heamocytometer slide. Cell viability studies were performed using Trypan blue dye exclusion assay.
  • rhodamine-dextran (Mol. wt. 70kDa) was encapsulated in the internal aqueous phase of the primary W/O emulsion or in the gelatin nanoparticles prior to formulating into the multiple emulsions.
  • the final concentration of rhodamine-dextran incorporated in both the formulations was 0.0025% (w/v).
  • the cells were washed with sterile phosphate buffered saline (PBS, pH 7.4) and the cover slip was removed from the wells and mounted on a clean glass slide. The samples were observed with an Olympus fluorescence microscope equipped with a BioQuant image analysis system.
  • PBS sterile phosphate buffered saline
  • Transfection efficiency was calculated as nanograms of EGFP present per milligram of total protein.
  • NiE formulation was better compared to ME or GNP formulations in causing gene transfection since NiE formulation offer more protection to plasmid DNA during formulation resulting in higher loading efficiency of plasmid DNA per ml of formulation as well as during intracellular transport of plasmid DNA in endosomal/lysozomal compartments.
  • mIL-10 plasmid transformed E. coli bacteria were obtained from Invivogen (San Diego, CA). Liquid agar broth was prepared by dissolving 35 g of LB Agar powder obtained from Sigma Aldrich (St. Louis, MO) in 1 L deionized water and sterilized by autoclaving at 121°C for 15 minutes. Lyophilized E. coli bacteria disc was suspended in liquid broth agar medium. The bacterial suspension was then streaked on solid agar plates and incubated for 12 hours at 37°C for growth of bacterial colonies.
  • electrophoresis was performed either with undigested plasmid or plasmid digested with Nhel and Ncol restriction enzymes obtained from New England Biolabs (Ipswich, MA).
  • RT-PCR Analysis for mIL-10 mRNA Expression Reverse transcriptase polymerase chain reaction is a technique for identification of gene expression at mRNA level.
  • IL-10 expression at the level of mRNA 200,000 J774A.1 cells were incubated with NiE, ME, GNP, Lipofectin® or naked IL-10 plasmid for 6, 12 and 24 hours. After completion of each time point, total cellular RNA was extracted from cells using High Pure RNA Isolation Kit (Roche, Indianapolis, IN) as per the kit protocol. Quantification of isolated RNA was performed using NanoDrop® 2000c instrument (Thermo-Fisher Scientific,
  • RNA fractions were converted to final PCR products using selective primers for IL-10 and beta-actin using One Step RT-PCR kit (Qiagen, Valencia, CA) as per kit manufacturer protocol.
  • the forward and reverse primer sequences used for conversion of IL-10 mRNA in to cDNA were 5 '-CCAGCCTTATCGGAAATGA-3 ' and 5'- TCTCACCCAGGGAATTCAAA-3' respectively.
  • the forward and reverse primer sequences used for conversion of ⁇ -actin mRNA into cDNA were 5 '-GTTACCAACTGGGACGACA-3 ' and 5'-TGGCCATCTCCTGCTCGAA-3' respectively.
  • the amount of template RNA added for each reaction was 1 ⁇ g.
  • PCR cycler settings for cDNA amplification are shown in Table 10. Final PCR products were run on 1.2% agarose E-gels (Invitrogen, CA) for visualization of cDNA bands using Kodak UV/NIR image station. Table 10: PCR cycler settings for RT-PCR experiments
  • Colorimetric sandwich ELISA plates for mIL-10 protein were purchased from R&D Systems (Minneapolis, MN). ELISA assay was performed as per the kit protocol. Briefly, 50 ⁇ of assay diluent and 50 ⁇ of standard, sample or control were added to different wells. After gentle mixing, plates were covered with a plate sealer and incubated for two hours. After two hours solution from each well was aspirated and wells were washed 5 times with IX wash buffer. After this step, 100 ⁇ of secondary antibody conjugated with horseradish peroxidase was added to each well and incubated for two hours. After two hours, again contents from each well was aspirated and wells were washed 5-times with IX wash buffer.
  • TNFa and ⁇ RT-PCR Analysis After completion of each time point, total cellular RNA was extracted from cells using High Pure RNA Isolation Kit (Roche, Germany) as per the kit protocol. Quantification of isolated RNA was performed using NanoDrop® 2000c instrument (Thermo-Scientific, Delaware). The isolated RNA fractions were converted to final PCR products using selective primers for TNFa and ILip and beta-actin using One Step RT- PCR kit (Qiagen, Valencia, CA) as per kit manufacturer protocol.
  • the forward and reverse primer sequences used for conversion of mRNA into cDNA for TNFa were 5'- CATGAGCACAGAAAGCATGATC-3 ' and 5 ' -CCTTCTCCAGCTGGAAGACT-3 ' respectively.
  • the forward and reverse primer sequences used for conversion of mRNA into cDNA for IL- ⁇ were 5 ' -GGCTGCTTCC AAACCTTTGA-3 ' and 5'-
  • ELISA assays were performed as per the kit protocols. Briefly, 50 ⁇ of assay diluent and 50 ⁇ of standard, sample or control were added to different wells in both plates. After gentle mixing, plates were covered with a plate sealer and incubated for two hours.
  • TNFa and ⁇ RT-PCR Analysis LPS triggered expression and release of proinflammatory cytokines was evaluated using RT-PCR analysis. At the end of 12 hour and 48 hour time points, levels of TNFa and IL1 ⁇ in cells pretreated with IL-10 gene containing formulations was lower compared to untreated cells. Among the different formulations tested, NiE formulation which was most efficient in causing mIL-10 gene transfection showed highest suppression of TNFa and ILip gene transcription followed by ME and GNP formulations.
  • TNFa and ⁇ Expression Analysis by ELISA ELISA for TNFa and ILip was performed in order to quantitate levels of these cytokines in cell culture supernatant at different time points following LPS stimulation on IL-10 transfected J774A.1 cells. The results ( Figures 29 and 30) showed highest suppression of both TNFa and ILip in cells treated with NiE followed by ME and GNP formulations. These results indicate that IL-10 plasmid delivered through NiE has therapeutic activity where it can suppress expression of pro inflammatory cytokines such as TNFa and ILi p.
  • plasmid EGFP-N1 or mIL-10 encapsulated in NiE had superior gene transfection efficiency compared to plasmid EGFP-N1 or mIL-10 encapsulated in ME or GNP or complexed with Lipofectin®, a cationic-lipid based transfection reagent.
  • mIL-10 delivered through NiE was capable of down regulating levels of proinflammatory cytokines TNFa and IL1 ⁇ in LPS stimulated J774A.1 cells.
  • Vitamin E uncouples joint destruction and clinical inflammation in a transgenic mouse model of rheumatoid arthritis. Arthritis Rheum. 46:522-532 (2002).

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US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
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CN113528634A (zh) 2012-08-14 2021-10-22 10X基因组学有限公司 微胶囊组合物及方法
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
AU2013302657B2 (en) 2012-08-15 2018-08-09 Tris Pharma, Inc. Methylphenidate extended release chewable tablet
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
KR20200140929A (ko) 2013-02-08 2020-12-16 10엑스 제노믹스, 인크. 폴리뉴클레오티드 바코드 생성
WO2014145242A1 (en) * 2013-03-15 2014-09-18 The Board Of Trustees Of The University Of Illinois Peptide-coated polymer carriers
CN113249435B (zh) 2014-06-26 2024-09-03 10X基因组学有限公司 分析来自单个细胞或细胞群体的核酸的方法
WO2016033556A1 (en) 2014-08-28 2016-03-03 Lipocine Inc. BIOAVAILABLE SOLID STATE (17-β)-HYDROXY-4-ANDROSTEN-3-ONE ESTERS
GB201420080D0 (en) * 2014-11-11 2014-12-24 Midatech Ltd And Q Chip Ltd Sustained release encapsulated nanoparticles
CA2966914A1 (en) * 2014-11-24 2016-06-02 The Procter & Gamble Company Systems for encapsulation of actives within droplets and other compartments
PL412787A1 (pl) 2015-06-22 2017-01-02 Magdalena Król Oparty na makrofagach celowany system dostarczania związków związanych z ferrytyną
US11590228B1 (en) 2015-09-08 2023-02-28 Tris Pharma, Inc Extended release amphetamine compositions
CN111032134B (zh) * 2017-06-30 2024-02-13 阿维塔斯有限公司 电喷雾导管
US11590081B1 (en) 2017-09-24 2023-02-28 Tris Pharma, Inc Extended release amphetamine tablets
CN111164068B (zh) 2018-08-21 2023-11-14 株式会社东芝 生物降解性化合物、脂质粒子、含有脂质粒子的组合物、及试剂盒

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479258B1 (en) 1995-12-07 2002-11-12 Diversa Corporation Non-stochastic generation of genetic vaccines
US6210707B1 (en) 1996-11-12 2001-04-03 The Regents Of The University Of California Methods of forming protein-linked lipidic microparticles, and compositions thereof
US6001311A (en) 1997-02-05 1999-12-14 Protogene Laboratories, Inc. Apparatus for diverse chemical synthesis using two-dimensional array
WO1999054459A2 (en) 1998-04-20 1999-10-28 Ribozyme Pharmaceuticals, Inc. Nucleic acid molecules with novel chemical compositions capable of modulating gene expression
DE10157046A1 (de) * 2001-11-18 2003-06-12 Novosom Ag Nano- und Mikrokapseln umfassend Reaktivpolymere
WO2004065578A2 (en) * 2003-01-14 2004-08-05 Chiron Corporation Microparticles comprising polynucleotide adsorbed on the surface of or entrapped within the microparticles
US20060204442A1 (en) * 2003-09-12 2006-09-14 Bankruptcy Estate Of Ferx, Inc. Magnetically targetable particles comprising magnetic components and biocompatible polymers for site-specific delivery of biologically active agents
CN101438252A (zh) * 2004-10-07 2009-05-20 爱莫里大学 多功能纳米粒子共轭体及其应用
US20060088533A1 (en) * 2004-10-08 2006-04-27 Charalabos Pothoulakis Methods of treating inflammatory bowel disease
AU2006235489A1 (en) * 2005-04-12 2006-10-19 Intradigm Corporation Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases
DK1757276T3 (da) * 2005-08-01 2008-02-04 Univ Muenchen Tech Fremgangsmåde til fremstilling af nano- og mikrokapsler af edderkoppesilkeprotein
EP1973608A1 (de) * 2005-12-14 2008-10-01 Cytos Biotechnology AG Immunstimulatorische nukleinsäure-packteilchen zur behandlung von überempfindlichkeit
CN100467114C (zh) * 2007-03-01 2009-03-11 上海交通大学 高分子微纳米囊的制备方法
WO2008115854A2 (en) * 2007-03-19 2008-09-25 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Multifunctional nanoparticles and compositions and methods of use thereof
DK2214646T3 (da) 2007-10-05 2021-10-04 Univ Wayne State Dendrimers for sustained release of compounds
AU2008314647B2 (en) * 2007-10-12 2013-03-21 Massachusetts Institute Of Technology Vaccine nanotechnology
KR20100037389A (ko) 2008-10-01 2010-04-09 연세대학교 산학협력단 다중 약물방출조절이 가능한 솔리드 마이크로구조체 및 이의 제조방법
WO2010148106A1 (en) * 2009-06-16 2010-12-23 Washington State University Research Foundation Systems and methods involving calcium phosphate based materials
WO2011026111A1 (en) 2009-08-31 2011-03-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Oral delivery of a vaccine to the large intestine to induce mucosal immunity
US20110229580A1 (en) * 2010-03-22 2011-09-22 Indian Institute of Technology Bombay, School of Biosciences and Bioengineering Compositions and methods for nano-in-micro particles

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US10500156B2 (en) 2019-12-10
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